1. Field of the Invention
The present invention relates to static diffusion cells useful in automated and manual diffusion sampling systems as well as assay methods that utilize diffusion sampling systems that include one or more diffusion cells according to the present invention. In particular, the present invention provides a static diffusion cell that includes a single chambered receptor compartment, which design reduces or eliminates the disadvantages associated with diffusion cells having multi-chambered receptor compartments and allows for improved sampling systems and assay methods.
2. Description of the Related Art
In vitro membrane diffusion systems with automated sampling are widely available for flow-through diffusion cells. However, applicants are presently aware of only two commercial systems that provide both static diffusion cells and automated sampling. Hanson Research Corp. of Chatsworth, Calif., sells the Hanson MicroettePlus™ Transdermal Diffusion System, and Logan Instruments Corp. of Somerset, N.J., sells the Logan System-902 and Logan System-912 Automated Transdermal Sampling Systems. Logan also plans to sell an upgraded system that includes a cell design similar to the 902-system and an XYZ robot for automatic sampling. Although, the diffusion systems available from Hanson Research and Logan Instruments exhibit differences in design, the systems available from both companies includes a large amount of small diameter tubing and pumps (peristaltic or syringe) that move fluids through multiple compartments within the systems.
As illustrated in FIG. 1, in the Logan system 10, tubing is part of the cell design. The receptor compartment consists of three chambers linked together by small diameter tubing. A diffusion membrane 11 is positioned on the diffusion chamber 12, which includes a stir bar 14, a sampling port 16 that allows introduction of a sampling probe 18, and a water jacket 20 with an inlet and an outlet that facilitate circulation of water around the diffusion chamber 12 to maintain the diffusion chamber 12 at a desired temperature, such as 37° C. A material or formulation to be evaluated 22 is placed over the diffusion membrane 11, and samples are collected from the collection chamber or flow cell 28 using a suitable collection apparatus. The third chamber 24 is used as a bubble trap. A peristaltic pump 26 continuously circulates the receptor medium 30 between the three chambers to maintain adequate mixing, except at sampling time. After receptor medium 30 flows out of the diffusion chamber 12 and flows through both the collection chamber 28 and the bubble trap 24, the receptor medium 30 returns to the diffusion chamber 12 through a media return 32. Process steps for the Logan system 10 include 1) taking a sample from the flow cell 28; 2) collecting the sample in an HPLC sample rack 34, wherein on-line injection is optional; 3) using gravity flow 40 from a replacement media bottle 38 to feed replacement media into a replacement media cell 36 obtained from the HPLC sample rack 34; and 4) adding the replacement media 36 at the flow cell 28 after sampling.
As illustrated in FIG. 2, in the Hanson MicroettePlus™ Transdermal Diffusion System 50, the diffusion cell 54 consists of a single chamber, but the input arm of the receptor chamber is connected by tubing to a syringe chamber 52 (Microette unit) and the output arm to a central sample collection chamber 56. Samples from the receptor chambers are collected in the central sample collection chamber 56 by positive displacement initiated by the syringe unit 52. Therefore, the MicroettePlus™ Transdermal Diffusion System 50 also utilizes multiple chambers interconnected by tubing.
The designs of the systems described above can make cell set-up, handling, and cleaning difficult and can lead to inaccurate experimental data. In particular, the relatively extensive use of tubing in the systems available from Logan Instruments Corp. and Hanson Research Corp. introduces several potential problems. For example, the tubing can clog or leak, and the use of tubing can result in variable or inaccurate calculations of cell volume. Perhaps even more problematic is binding of media constituents, such as the material to be assayed, to the tubing or leaching of chemicals from the tubing into the receptor medium. Both the binding of materials from the receptor medium to the tubing and the leaching of materials from the tubing into the receptor medium can lead to an inaccurate assay of the amount or type of materials that diffuse through the diffusion membrane and into the receptor medium.
Further, systems designed according to those available from Logan Instruments Corp. and Hanson Research Corp. permit the accumulation of air bubbles under the diffusion membrane even after receptor medium has been degassed. Accumulation of air bubbles under the diffusion membrane causes a reduction in the area of the diffusion membrane in contact with the receptor medium, thereby reducing the effective diffusion area. Such a reduction in diffusion area can, in turn, result in inaccurate experimental data. However, removing air bubbles from the systems available from Logan Instruments Corp. and Hanson Research Corp. system is tedious at set-up and very difficult or even unattainable once a diffusion experiment has started. Accordingly, when these systems are used, they typically require supervision to ensure that air bubbles do not accumulate under the diffusion membrane, which supervision defeats, at least in part, the purpose of having an automated sampling process.